Abstract

T cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocytic choriomeningitis virus infection in mice, and PD-1hi cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, hepatitis C virus, and hepatitis B virus. However, it is not known if PD-1hi cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function, and gene expression profiles of PD-1hi versus PD-1lo CD8 T cells in the peripheral blood of healthy human adults as follows: 1) the percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans, and PD-1 was expressed by the memory CD8 T cells; 2) PD-1hi CD8 T cells in healthy humans did not significantly correlate with the PD-1hi exhausted gene signature of HIV-specific human CD8 T cells or chronic lymphocytic choriomeningitis virus-specific CD8 T cells from mice; 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults; 4) PD-1 was expressed by the effector memory compared with terminally differentiated effector CD8 T cells; and 5) finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed. In conclusion, our study shows that most PD-1hi CD8 T cells in healthy adult humans are effector memory cells rather than exhausted cells.

CD8 T cells are a critical component of the immune system and are responsible for killing virus-infected cells and control of persistent and reactivating viruses. However, persistent antigenic stimulation leads to CD8 T cell exhaustion, characterized by the induction of a hypoproliferative state and loss of the ability to produce antiviral cytokines (1). Reversing CD8 T cell exhaustion could provide a promising therapeutic approach for enhancing natural immunological control over chronic viral infections.

Programmed death-1 (PD-1) is a member of the CD28 family of immune modulators (2–4). It is upregulated on CD8 and CD4 T cells upon activation. PD-1 binds to its ligands PD-L1 (B7-H1) or PD-L2 (B7-DC). Ligation of PD-1 results in dephosphorylation of signaling molecules downstream of the TCR, thus dampening T cell sensitivity to antigenic stimulation. Significant evidence suggests that this pathway inhibits T cell responses upon persistent antigenic stimulation (5–7). It has been shown that immunoreceptor tyrosine-based switch motif of PD-1 cytoplasmic tail recruits tyrosine phosphatases, Src homology region 2 domain-containing phosphatase-1, and -2, which in turn interferes with proximal TCR signaling pathways to affect T cell functions (8). PD-1–deficient mice, when crossed to the NOD background, experience more rapid and severe diabetes (9–12). PD-L1−/− mice die of excessive T cell immunopathology following chronic lymphocytic choriomeningitis virus (LCMV) infection (13). We have shown that PD-1 regulates T cell dysfunction during chronic LCMV infection in mice and demonstrated that in vivo blockade of PD-1–PD-L1 interactions: 1) restores effector functions of exhausted CD8 T cells; and 2) leads to substantial reduction in virus replication (13). These findings were extended to HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) infections in humans and SIV infection in macaques, indicating that PD-1 overexpression on T cells plays an important role in these infections (14–20). These data suggest that abrogation of the PD-1 inhibitory pathway may contribute to successful treatment of life-threatening chronic infections in humans.

In addition to chronic viral infections, PD-1 inhibitory pathway plays an important role in tumors. Expression of PD-L1 is reported on a variety of human tumors. Expression of PD-L1 by tumor cells correlates with a very poor prognosis, suggesting that cancer cells purloin this inhibitory pathway to evade the host immune response (21, 22). Recent studies also show that the tumor microenvironment may play a role in the induction and maintenance of PD-1 expression on tumor-reactive T cells and that expression of PD-1 on tumor-infiltrating lymphocytes impairs the antitumor immune responses in humans (23).

Although PD-1 expression on virus-specific CD8 T cells of chronically infected patients is well described, little is known about its pattern expression on CD8 T cells of healthy human adults. Although PD-1 contributes to functional defects of memory CD8 T cells, deficiencies in the PD-1 are associated with autoimmune diseases (5, 10, 24). PD-1 is transiently expressed on activated T cells, and frequent TCR stimulation is required to maintain PD-1 expression (13, 25, 26). However, the proportion of activated versus exhausted cells expressing PD-1 is not known. Hence it is also essential to delineate when PD-1 expression is the signal of physiologic regulatory mechanisms of activated cells, a marker of exhausted cells, or a mediator of functional exhaustion.

For this reason, we examined the levels of PD-1 expression on the resting CD8 T cells from healthy individuals. We found that PD-1 is expressed by ∼60% of memory CD8 T cells in the peripheral blood cells of healthy humans. First, we analyzed the gene expression profile of PD-1hi CD8 T cells in the peripheral blood cells of healthy human adults in comparison with PD-1lo and naive CD8 T cells. We found that the gene expression profiles of PD-1hi and PD-1lo CD8 T cells were closely related. Then we compared gene expression profiles of the PD-1hi CD8 T cell subset of healthy humans with PD-1hi exhausted signatures of HIV-specific human CD8 T cells or LCMV-specific CD8 T cells from chronically (LCMV clone-13 strain) infected mice. We found that signatures of genes characteristic of exhausted CD8 T cells were not enriched in the PD-1hi CD8 T cell subset in healthy humans. Next, we compared the gene expression profile of PD-1hi CD8 T cells with their phenotypic and functional characteristics. Phenotypic analysis also revealed that the majority of PD-1–expressing cells do not show exhausted characteristics. Both the gene array and phenotypic analysis demonstrated that the majority of PD-1 is expressed by the effector memory (TEM) compared with terminally differentiated effector memory RA T cells (TEMRA). Interestingly, we observed a relationship between CD45RA and PD-1 expression that CD45RA and PD-1 expression levels were inversely related. CD8 T cells with the lowest CD45RA expression contained the highest proportion of PD-1hi cells and CD8 T cells with the highest CD45RA expression contained the lowest proportion of PD-1hi cells. Our study suggests that expression of PD-1 may be the signal of regulatory mechanisms of both activated and exhausted cells in healthy human adults, and PD-1 expression should not be regarded as a definitive marker for exhausted cells.

Materials and Methods

Blood samples

Peripheral blood samples were obtained from healthy human adults (25–40 y of age). PBMC were isolated from the blood using vacutainer cell preparation tubes (BD Biosciences, San Diego, CA). RBCs were lysed by incubation with ACK lysis buffer (Life Technologies) for 2 min at room temperature. After extensive washing, the PBMC were resuspended in RPMI 1640 medium containing 10% FCS supplemented with penicillin, streptomycin, and l-glutamine as described (27). All subjects were recruited at the Emory University Vaccine Center and gave informed consent for the study.

Isolation, amplification, and labeling of mRNA for gene array

PD-1hi, PD-1lo, and naive human CD8+CD3+ T cells were FACS sorted, and total RNA was isolated from cells in TRIzol (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized using the SuperScript Choice cDNA synthesis kit (Invitrogen) and an oligo(dT) primer containing a T7 promoter. The T7 MEGAscript kit (Ambion) was used to amplify cRNA from the cDNA. The cRNA was reverse transcribed with biotinylated nucleotides using the Enzo BioArray High Yield RNA transcript labeling kit (Enzo Life Sciences) in the second round of cRNA synthesis, fragmented, and hybridized on Affymetrix Human genome U133 Plus 2.0 arrays (Affymetrix) at the Vanderbilt University Microarray Shared Resource, according to the manufacturer’s protocols.

Microarray analysis

Gene expression data were summarized with RMA and merged according to each cell type (PD-1hi, PD-1lo, and naive). Each cell-type group was then compared by t test (p < 0.05). The genes with low fold change values (1.6 cutoff) and normalized intensity values <100 in both compared conditions were further removed to reduce false positivity. Each group’s signature genes were then cross examined to extract the unique gene sets among PD-1hi, PD-1lo, and naive CD8 T cell subsets. These gene sets were examined through downstream analysis methods. Using the Database for Annotation, Visualization, and Integrated Discovery, we categorized the biological processes in which the genes were involved, their molecular function, or related pathways.

We have done two types of analyses with the data set as described (28–34). 1) Comparative marker analysis: to test differentially expressed genes (DEGs) among PD-1hi and PD-1lo cells. For direct comparison of gene expression profiles of PD-1hi and PD-1lo cells, PD-1lo cell groups were used as controls for baseline transformation of PD-1hi cell groups. The t test with p value of 0.05 and fold change value of 1.6 with no correction was used. 2) Gene set enrichment analysis (GSEA): it is a method for determining whether a rank-ordered list of genes for a particular comparison of interest is enriched in genes derived from an independently generated gene set (e.g., PD-1hi and PD-1lo cells). GSEA provides an enrichment score that measures the degree of enrichment of a given gene set at the top (highly correlated) or bottom (anticorrelated) of the second, rank-ordered data set (28–33). A nominal p value is used to assess the significance of the enrichment score.

GSEA was performed with the following three different sets of genes: 1) exhausted gene sets, to examine if exhausted genes were enriched. These gene sets were those upregulated in LCMV exhausted (versus naive, effector, memory) and HIV progressors (versus elites) (32); 2) KIR gene set (Table II); and 3) naive, TEM, and TEMRA subsets (34–37).

Intracellular cytokine analysis

For intracellular cytokine staining, fresh PBMC were stimulated in vitro for 6 h with plate-bound anti-CD3/CD28 or PMA/ionomycin in 96-well round-bottom plates in the presence of brefeldin A (1 μl/ml). The cells were washed once with FACS buffer and stained with relevant T cell markers for 30 min at room temperature. Then the cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences), washed twice with permeabilization buffer, and stained for cytokines using anti–IFN-γ, TNF-α, IL-2, or MIP-1β. After washing, the cells were acquired on an FACSCalibur (BD Biosciences) or LSR II flow cytometer (BD Biosciences) using FACSDiva software (BD Immunocytometry Systems). Flow cytometry analysis was performed using FlowJo software (Tree Star, Ashland, OR).

Statistical analysis

Comparison between groups (p value) was calculated using the Student t test and Wilcoxon t test. All statistical analysis was performed using the GraphPad Prism program (GraphPad).

Genome-wide microarray analysis of PD-1hi and PD-1lo CD8 T cells of healthy adults. A, Representative data showing sorting strategy of PD-1hi, PD-1lo, and naive subpopulations among CCR7loCD8+/CD3+ T cells. Expression level of mRNA was measured by microarray analysis of sorted PD-1hi, PD-1lo, and naive cells. B, Total number of transcripts differentially expressed (up- or downregulated; 1.6-fold cutoff) between the indicated comparison groups is shown. C, Relative intensities of the DEGs in any comparison with a 1.6-fold cutoff in naive, PD-1hi, and PD-1lo populations are plotted as heat maps to depict the relationship among these three populations. Expression level of each gene is represented by the number of standard deviations above (red) or below (green) the average value for that gene across all samples. D, Heat map showing relative intensities of the DEGs between PD-1hi and PD-1lo populations with a 1.6-fold cutoff. E, GSEA showing enriched profile of KIR gene set in PD-1lo cells.

A list of genes that are up- and downregulated on PD-1hi cells is presented in Tables I and II. Compared to PD-1lo cells, PD-1hi cells upregulated expression of genes encoding costimulatory (CD28 and CD27) and coinhibitory (CTLA-4) receptors, effector molecule (granzyme K), cell signaling and proliferation molecules (PASK and E2F3), and homing receptors (CXCR6, CXCR4, and CCR5). In addition, PD-1hi cells also show downregulation of some positive TCR signaling molecules such as TYROBP, LAT2, LYN, and VAV3.

One of the striking observations in this study is that all of the killer cell Ig-like receptors (KIRs) and KLRs were completely downregulated on PD-1hi cells (Table II). We tested enrichment of genes encoding all of the KIRs on PD-1lo cells using GSEA as described in the Materials and Methods (28–33). For this, we compiled a set of KIR genes from the Database for Annotation, Visualization, and Integrated Discovery. Then we determined if this curated KIR gene set was enriched in genes that were increased in expression in PD-1hi versus PD-1lo CD8 T cells of healthy subjects. As shown in Fig. 2E, we confirmed highly significant (p < 0.001) enrichment of genes encoding KIRs on PD-1lo cells (Fig. 2E).

We next tested whether PD-1hi cells showed global similarity to exhausted cells by comparing gene expression profiles from PD-1hi CD8 T cells to previously published microarray data from exhausted, virus-specific CD8 T cells in humans and LCMV mouse models (29, 32) (Fig. 3). We identified sets of genes upregulated in HIV-specific CD8 T cells from chronic progressors compared with HIV-specific CD8 T cells from controllers (32) and queried our PD-1hi versus PD-1lo data set for global enrichment of the exhausted progressor signature (28–32). If PD-1hi cells of healthy subjects represented a more exhausted population, one would predict that the exhausted gene set would be more enriched in the PD-1hi than PD-1lo subset. However, GSEA showed no significant enrichment of exhausted gene signature in the PD-1hi subset compared with PD-1lo subset (Fig. 3). We repeated this analysis with a set of genes upregulated in exhausted LCMV-specific CD8 T cells in mice and found no enrichment of exhausted signature genes in our PD-1hi subset from healthy individuals (Fig. 3). These findings suggest that PD-1hi CD8 T cells from healthy individuals do not share a global gene expression pattern with exhausted CD8 T cells in chronic viral infection.

We then examined the relationship between T cell differentiation state and PD-1 expression. For this, we identified sets of genes that were characteristic of human memory CD8 T cell subsets—naive, TEM, and TEMRA—from the published data (34). For each class of samples, we identified unique sets of upregulated genes; for example, naive unique genes are those specifically upregulated (p < 0.05) in the naive subset compared with TEM and TEMRA. Similarly, we obtained specifically expressed unique genes by TEM and TEMRA subsets. We found that 681 of naive, 53 of TEM, and 226 of TEMRA were unique genes (Fig. 4). Then we created three custom modules for each subset to run GSEA to test how these unique gene sets are enriched in our data set comparing PD-1hi versus PD-1lo data sets. As expected, the naive gene set did not enrich in either PD-1hi or PD-1lo data sets (Fig. 4). In contrast, we found significant enrichment of the TEM gene set in PD-1hi cells (p = 0.035) and the TEMRA gene set in PD-1lo cells (p = 0.005) (Fig. 4).

Phenotypic analysis of PD-1–expressing CD8 T cells from the blood of healthy adults

Recent evidence from chronic LCMV infection in mice and HIV, HBV, or HCV infections in humans indicates that upregulation of PD-1 correlates with impaired CD8 T cell phenotypic and functional properties (14–19). However, in our study, microarray analysis of PD-1hi CD8 T cells from healthy adults demonstrated that these cells did not exhibit exhausted characteristics of PD-1hi CD8 T cells of LCMV or HIV. Hence, we carried out phenotypic analysis to correlate the results of microarray analysis. The gating strategy of PD-1hi, PD-1lo, and naive CD8 T cells from a representative subject is shown in Fig. 5A. We compared the phenotypic characteristics of CCR7loPD-1hi or CCR7loPD-1lo in the context of naive (CD45RA+CCR7+) cells (Fig. 5B).

Phenotypic analysis of PD-1hi, PD-1lo, and naive CD8 T cells of healthy adults. A, An example of gating strategy of PD-1hi and PD-1lo CD8+/CD3+ T cells that are CCR7 negative is shown. Naive cells (CD45RA+/CCR7+) that do not express PD-1 are shown. B, Flow cytometry analysis showing percent expression and mean fluorescence intensity (MFI) of indicated markers on PD-1hi and PD-1lo CCR7loCD8+/CD3+ T cells in comparison with naive CD8+/CD3+ T cells of the corresponding donors are shown (n = 5–15). The percentage of CD8 T cells expressing the relevant receptor for each individual (open circle) and the group mean (horizontal line) are shown.

We analyzed coexpression of PD-1 and several T cell differentiation markers (26, 35–40). CD27 and CD28 are costimulatory receptors involved, respectively, in the generation of Ag-primed cells and the regulation of T cell activation (39, 40). In this study, we observed that PD-1hi CD8 T cells in healthy adults expressed both CD28 and CD27 at higher levels compared with PD-1lo CD8 T cells.

It has been well demonstrated that CD8 T cells downregulate cytokine receptor CD127 (IL-7Rα) expression as they differentiate following Ag encounter and selectively re-express on a subset destined to form precursors of the memory pool (41–43). We observed substantial expression of CD127 on memory cells, though not as high as by naive cells. Among the memory cells, PD-1hi cells showed higher expression levels of CD127 compared with PD-1lo cells. It was reported that memory cells retain expression of activation marker CCR5, although not as high as activated cells (38). Our study also showed that CD8 T cells of healthy humans expressed CCR5; however, there was no difference in expression levels among PD-1hi and PD-1lo CD8 T cells.

Cytolytic potential of the effector T cells is characterized by the expression of granzyme B and perforin. Our results showed that both PD-1hi and PD-1lo CD8 T cells of healthy humans express similar levels of granzyme B. However, perforin expression was lower among PD-1hi CD8 T cells compared with PD-1lo CD8 T cells of healthy humans. It has been shown that resting cells do not express Ki-67, whereas cycling or very recently divided T cells upregulated Ki-67 expression (44). Consistent with this, PD-1hi or PD-1lo CD8 T cells of healthy subjects do not express Ki-67. In addition, the absence of Ki-67 expression clearly correlated with the lack of CD38 and HLA-DR expression on PD-1hi or PD-1lo CD8 T cells in our study.

KLRG-1 is an NK cell receptor expressed by T cells that exhibit terminal effector properties (45, 46). Our results showed that both PD-1hi and PD-1lo cells express similar levels of KLRG-1. The expression of antiapoptotic protein Bcl-2 was expressed at lower levels on PD-1hi cells compared with PD-1lo cells, suggesting that PD-1hi cells are prone to apoptosis. Naive cells showed expression of higher levels of Bcl-2. Opposing expression of PD-1 and CD57 during CD8 T cell maturation was described, with high-level PD-1 expression having an impact upon ex vivo sensitivity to apoptosis (47). However, both PD-1hi and PD-1lo CD8 T cells of healthy humans express similar levels of CD57. In addition, PD-1 expression was linked to a proapoptotic phenotype characterized by high expression of CD95/Fas (48). Consistent with this, our study showed positive correlation between PD-1 and CD95 expression among CD8 T cells of healthy humans.

In mice, naive CD8 T cells express moderate levels of α4β7 integrin, which is required for homing to Peyer’s patches (49), whereas recently activated effector CD8 T cells express high levels of α4β7 and migrate to effector sites including small intestinal epithelium (50). We found that α4β7 expression delineated two populations among CCR7lo CD8 T cells in healthy adult human PBMC: a subset that lacked α4β7 expression and another that expressed similar levels as naive CD8 T cells. PD-1hi CD8 T cells were enriched for α4β7+ cells, suggesting that PD-1hi cells may have different trafficking patterns than PD-1lo CD8 T cells.

It has been shown that naive cells express CD45RA, its expression is downregulated during effector differentiation, and it is re-expressed on resting memory cells in the absence of further restimulation (38, 51). Our results showed that PD-1hi CD8 T cells downregulate CD45RA compared with PD-1lo CD8 T cells of healthy humans.

Coexpression of PD-1 and other inhibitory receptors on the CD8 T cells of healthy human adults

Expression of PD-1 does not affect the ability of CD8 T cells in healthy adults to secrete cytokines

High-level expression of PD-1 was associated with a diminished ability of LCMV-specific CD8 T cells to secrete cytokines in chronically infected mice (13). However, human HIV-specific CD8 T cells showed that PD-1 expression was not directly associated with impaired cytokine secretion (15). In this study, we tested if stimulation of PBMC with anti-CD3/CD28 shows any difference in the secretion (or lack thereof) of IFN-γ, TNF-α, IL-2, or MIP-1β by PD-1hi or PD-1lo CD8 T cells. We stimulated total CD8 T cells with anti-CD3/CD28 for 6 h at 37°C and then assessed cytokine secretion by intracellular cytokine staining. We did not observe any difference in production of IFN-γ, TNF-α, IL-2, or MIP-1β between PD-1hi and PD-1lo CD8 T cells (Fig. 7). Our results indicate that PD-1 expression in healthy adults did not affect the ability of CD8 T cells to secrete cytokines.

No direct association between PD-1 expression and cytokine production by CD8 T cells of healthy adults. PBLs from healthy individuals were stimulated with anti-CD3/CD28 for 6 h at 37°C, and intracellular staining of IFN-γ, TNF-α, IL-2, and MIP-1β was done. Representative data from one subject (of 16 healthy adults tested) showing percentage of IFN-γ, TNF-α, IL-2, and MIP-1β secretion by naive, PD-1hi, and PD-1lo CD8+/CD3+ T cell subsets.

PD-1 is expressed by the majority of TEM CD8 cells in the blood of healthy humans

Gene expression analysis showed that the majority of PD-1 was expressed by TEM compared with TEMRA (Fig. 4). To confirm this, we assigned PD-1–expressing CD3+/CD8+ T cells into four subsets based on expression of CCR7, lymphoid tissue homing receptor, and CD45RA, a transmembrane tyrosine phosphatase: 1) naive (CCR7+CD45RA+); 2) central memory (TCM: CCR7+CD45RA−); 3) effector memory (TEM: CCR7−CD45RA−); and 4) terminal effectors (TEMRA: CCR7−CD45RA+) (35–37). As shown in Fig. 8A and 8B, naive cells did not express PD-1. In contrast, 60% of TEM expressed PD-1, and approximately one third of TEMRA express PD-1. We also observed about one fourth of TCM expressed PD-1.

To further examine PD-1 levels among TEM and TEMRA CD8 T cells, we further subdivided CCR7lo CD8 T cells into four populations based on the levels of CD45RA expression. We observed an interesting relationship between CD45RA and PD-1 expression in that their expression levels are inversely related (Fig. 8C–E). Specifically, CD8 T cells with the lowest CD45RA expression contained the highest proportion of PD-1+ cells, and CD8 T cells with the highest CD45RA expression contained the lowest proportion of PD-1+ cells. Overall, these data demonstrate that the majority of PD-1 is expressed by the TEM CD8 T cells in the peripheral blood cells of healthy humans.

PD-1 is expressed at low to moderate levels on cytomegalovirus- and EBV-specific CD8 T cells

PD-1 is expressed at low to intermediate levels by CD8 T cells specific for common chronic viral infections (EBV and CMV). A, Representative PD-1 staining on EBV, CMV, influenza, and vaccinia virus-specific CD8+/CD3+ T cells. Peptide epitopes are abbreviated to the first three amino acids. Percentage and MFI of PD-1 expression among tetramer+ cells are indicated. B, Summary of percentage and MFI of PD-1 expression on CMV- and EBV-specific CD8 T cells in comparison with influenza and vaccinia virus-specific CD8 T cells from healthy subjects is shown. Individual results from each subject (individual symbols) and group mean (horizontal line) are shown.

Discussion

The T cell response is regulated by balance between costimulatory and coinhibitory signals. T cell exhaustion was initially characterized in LCMV model in mice, and these findings were extended to human chronic infections. All of these data demonstrate that PD-1 is highly expressed on chronic virus-specific CD8 T cells, and its upregulation is correlated with high viral load and associated with a high level of viremia. In contrast to continuous productive replication of HIV, HBV, or HCV, common human herpesviruses such as EBV and CMV are characterized by a stable cell–virus relationship and CD8 T cell-mediated control. PD-1 is not only expressed on exhausted T cells but also transiently expressed on activated T cells. The mechanism of PD-1 regulation in activated and exhausted cells is still poorly defined. In this study, we performed an in-depth analysis of PD-1hi and PD-1lo CD8 T cells to understand whether PD-1hi cells in healthy adult humans represent exhausted cells.

We found that the ratio of naive and memory CD8 T cells vary widely among healthy adults. Also, PD-1 expression level in memory cells vary among these subjects from 40 to 80% (Fig. 1B). This variation in PD-1 expression could be because of the flux occurring between maturation steps depending on the time of viral reactivation or in response to changes in viral load. When the Ag is low, cells are PD-1lo. During recrudescence, some of these PD-1lo cells are recruited back to effector phenotype, resulting in upregulation of PD-1 expression. Thus, in response to fluctuations in the Ag load, change in the distribution of PD-1hi CD8 T cells across different subsets can occur.

We compared gene expression profiles of PD-1hi CD8 T cell subset of healthy humans with PD-1hi exhausted signatures of LCMV- or HIV-specific CD8 T cells. We found that genes characteristic of functionally exhausted CD8 T cells were not enriched in PD-1hi CD8 T cells in healthy humans. This suggests that the PD-1hi CD8 T cell subset in the peripheral blood of healthy human subjects does not share global similarity in gene expression to exhausted T cells. However, the PD-1hi compartment consists of a heterogeneous set of Ag-experienced cells, and we therefore cannot exclude the possibility that a minority of this population may share features of exhaustion.

The phenotypic analysis of PD-1hi CD8 T cells of healthy human adults does not exhibit exhausted characteristics. For example, PD-1hi CD8 T cell responses to chronic viral infections, such as HIV, in humans report progressive downregulation of CD127 and CD28 (14–19). This phenotype is likely a direct consequence of chronic stimulation in a situation of high Ag load (63, 64). In contrast, healthy adults carry asymptomatic EBV and CMV that are characterized by low level of stimulation, Ag load, and recurrence, which do not downregulate CD127 and CD28 but maintain PD-1 at low to intermediate levels (Fig. 9). In addition, our data show that expression of PD-1 had no direct effect upon the ability of human CD8 T cells to produce cytokines.

Gene expression profiles using gene sets representative of naive, TEM, and TEMRA show that the majority of PD-1 is expressed by TEM compared with TEMRA CD8 T cells. Recent studies emphasized the heterogeneity of effector and memory cell populations with the description of multiple cellular subsets based on phenotype and function. Ag-experienced human CD8+ T cells were delineated into naive, TCM, TEM, and TEMRA subsets based on the patterns of CD45RA and CCR7 expression (35–37). Previous studies report that TEMRA phenotype is associated with terminal effector cells with little proliferative capacity, sensitive to apoptosis, and associated with senescence. In contrast, this study shows that PD-1 is predominantly expressed by TEM rather than TEMRA. In fact, we recently showed that yellow fever virus-specific memory CD8 T cells had a TEMRA phenotype (38). Hence, it is also possible that the low-level expression of PD-1 by TEMRA could be associated with the presence of memory CD8 T cell populations that do not express PD-1.

In conclusion, our study shows that most of the PD-1–expressing cells in healthy human adults do not represent characteristics of exhausted cells.

Disclosures

G.J.F. draws royalties from patents regarding PD-1. The authors have no other financial conflicts of interest.

Acknowledgments

We thank R. Karaffa, S. Durham, and S. Mertens for assistance with FACS and members of the Ahmed laboratory for helpful discussions.

Footnotes

This work was supported by National Institutes of Health Grant P01 AI080192-01 (to R.A.) and Grand Challenges in Global Health Initiative Grant 05GCGH0 (to R.A.).

The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession number GSE26495.